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Abstract:

Provided herein are methods of treating diseases mediated by PIM-1 and/or
PIM-2 and/or PIM-3 kinases by administering to a mammal in need thereof a
therapeutically effective amount of a compound of Formula I:
##STR00001##
in which B, R1, R1a, R2, R3, R4, R5,
R6, R7, R10 and R11 have the meanings given in the
specification.

Description:

[0001] This application is a Continuation of U.S. application Ser. No.
13/059,881, filed Feb. 18, 2011, which is a 371 filing of
PCT/US2009/054198 filed Aug. 18, 2009, which claims priority to U.S.
Provisional Application Ser. No. 61/089,958, filed Aug. 19, 2008, each of
which is incorporated herein by reference in its entirety.

[0002] The present invention relates to novel compounds, to pharmaceutical
compositions comprising the compounds, to a process for making the
compounds and to the use of the compounds in therapy. More particularly,
it relates to certain triazolopyridine compounds useful in the treatment
and prevention of diseases which can be treated with a PIM kinase
inhibitor, including diseases mediated by PIM kinases. Particular
compounds of this invention have been found to be inhibitors of PIM-1
and/or PIM-2 and/or PIM-3.

[0003] Protein kinases constitute a family of structurally related enzymes
that are responsible for the control of a vast array of cellular
processes.

[0006] Recently, it has been discovered that PIM-1 is up-regulated by
Flt-3 and may play an important role in Flt-3 mediated cell survival
(Kim, K. T. et al Neoplasia, 2005, 105(4): 1759-1767). Since Flt-3 itself
is implicated in leukemias like AML, additional knockdown of PIM-1 may be
a useful approach to treating leukemias driven by Flt-3 or various
mutations. Accordingly, PIM-1 inhibitors may be useful as therapeutic
agents for a variety of cancers such as hematological cancers.

[0009] Based upon the direct involvement of the PIM kinases in a wide
variety of cancers downstream of STAT3/5 activation, it is expected that
inhibition of the PIM kinases will result in inhibition of proliferation
and survival of multiple cancer cell types. This would then be expected
to provide a therapeutic benefit to cancer patients with a variety of
cancers (both solid tumor and hematologic settings), as well as other
conditions that are mediated by PIM kinase signaling.

[0010] In addition to the malignant cells detailed above, PIM kinases are
also expressed in hematopoietically-derived cell lines and
hematopoietically-derived primary cells including cells of the immune
system such as B cells, T cells, monocytes, macrophages, eosinophils,
basophils, and dendritic cells. Expression of PIM kinases can be induced,
for example, by cytokines which utilize Jak/Stat signaling, such as IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-12, IL-15, GM-CSF, IFNα,
IFNγ, erythropoietin, thrombopoietin, and prolactin, and the
generation, differentiation, maintenance and activation of
hematopoietically-derived cells is dependent on these cytokines.
Moreover, PIM proteins have been shown to be required for the efficient
proliferation of peripheral T cells mediated by T-cell receptor and IL-2
signaling (Mikkers, et al., Mol. Cell Biol., 2004, 6104). Although the
exact mechanism of action of PIM kinases in an immunological setting has
yet to be fully defined, they have been reported to phosphorylate a
number of substrates involved in cellular proliferation, differentiation,
and survival (Bullock et al., J. Biol. Chem., 2005 280:41675; Chen et
al., PNAS 2002 99:2175; Dautry et al. J. Biol. Chem. 1998 263:17615).

[0011] Chronic and acute inflammatory and autoimmune diseases are
associated with the overproduction of pro-inflammatory cytokines and
activation of immune cells against the body's own tissues. However, many
of these diseases are not adequately treated by current therapies and/or
these therapies have significant side effects/risks.

[0012] A particular example of an autoimmune disease is multiple sclerosis
(MS). MS is a progressive central nervous system (CNS) inflammatory
autoimmune disease wherein the immune system mounts responses against CNS
components. The resulting damage to axons and nerves leads to progressive
neurological impairment and significant disability. MS affects over 2.5
million people worldwide (www.nationalmssociety.org); however many
current therapies are only moderately effective and have questionable
risk factors

[0013] A need therefore remains for compounds and methods for treating
autoimmune and inflammatory diseases.

[0015] It has now been found that certain [1,2,4]triazole[4,3-a]pyridine
compounds bearing a quinolinyl group at the 3 position of the
triazolopyridine ring are inhibitors of PIM kinases, in particular PIM-1
and/or PIM-2 and/or PIM-3 kinases, which are useful for treating diseases
such as cancers and inflammatory diseases. In addition, compounds of the
invention may be useful for treating immune cell-associated diseases and
disorders, such as inflammatory and autoimmune diseases.

[0016] Accordingly, provided is a compound of the general Formula I:

##STR00002##

[0017] or a pharmaceutically acceptable salt thereof, wherein:

[0018] R10 and R11 together with the N to which they are
attached form a 4-8 membered heterocyclic ring optionally having an
additional ring heteroatom selected from N and O, wherein the
heterocyclic ring is optionally substituted with one or more R9
groups;

[0041] The phrase "R10 and R11 together with the N to which they
are attached form a 4-8 membered heterocyclic ring" refers to a group
having the formula --NR10R11 representing a 4-8 membered
saturated heterocyclic radical having at least one ring nitrogen atom.
The heterocyclic ring optionally has an additional ring heteroatom
selected from N and O, the remaining ring atoms being carbon.

[0042] In certain embodiments, the --NR10R11 group forms a 5-7
membered heterocyclic ring. In certain embodiments, the heterocyclic ring
is substituted with one or more R9 groups, for example 1-4 R9
groups, and as a further example 1-2 R9 groups.

[0045] In certain embodiments, R9 is (1-6C)alkyl. Examples include
methyl, ethyl, and propyl. A particular example is Me.

[0046] In certain embodiments, R9 is NRfRg. Examples
include groups where Rf is H or Me and Rg is H, methyl, ethyl,
propyl, isopropyl, butyl, or isobutyl. Particular values of R9 when
represented by NRfRg include NH2.

[0047] In certain embodiments, R9 is (1-6C alkyl)NRhRi.
Examples include groups where Rh is H and Ri is H or alkyl.
Particular values of R9 when represented by -(1-6C
alkyl)NRhRi include CH2NRhRi, for example
CH2NH2.

[0048] In certain embodiments, R9 is ORj. Examples include
groups where Rj is H or (1-6C) alkyl, for example methyl. A
particular example is OH.

[0049] In certain embodiments, R9 is (1-6C alkyl)ORk. Examples
include groups where Rk is H. A particular example is CH2OH.

[0050] In certain embodiments, R9 is C(O)NRmRn. Examples
include groups where Rm and Rn are independently H or (1-6C)
alkyl, for example methyl. A particular example is C(O)NH2.

[0051] In certain embodiments, R9 is C(O)O(1-6C alkyl). Examples
include CO2Me and CO2Et.

[0053] In certain embodiments, the heterocyclic ring represented by
--NR10R11 is a 5-7 membered heterocyclic ring which is
unsubstituted or substituted with one or more R9 groups
independently selected from Me, NH2, CH2NH2, OH,
CH2OH, C(O)OMe, C(O)NH2 and CH2NHCO2t-Bu. In certain
embodiments, the heterocyclic ring represented by NR10R11 is
substituted with one or two of said R9 groups.

[0054] Particular embodiments of heterocyclic rings represented by
--NR10R11 include the structures:

##STR00003##

[0055] In certain embodiments, B is H.

[0056] In certain embodiments, B is ORa.

[0057] Examples of B when represented by ORa include groups wherein
Ra is (1-6C)alkyl. Particular values include OMe, OEt and
O-(isobutyl). Particular mention is made of OMe.

[0058] Examples of B when represented by ORa include groups wherein
Ra is -(1-6C alkyl)-O-(1-6C alkyl). Particular values of ORa
include --OCH2CH2OMe and --OCH2CH2CH2OMe.
Particular mention is made of --OCH2CH2OMe.

[0059] Examples of B when represented by ORa include groups wherein
Ra is -(1-6C alkyl)-O-(3-6C cycloalkyl). A particular of ORa
includes --OCH2CH2O(cyclopropyl).

[0060] In certain embodiments, B is (1-6C alkyl)NRbRc. In
certain embodiments, Rb is H. In certain embodiments, Rc is H.
In other embodiments, Rc is (1-6C)alkyl. Examples include groups
having the formula CH2NRbRc, for example CH2NHEt and
CH2NH2.

[0061] In certain embodiments, B is (1-6C alkyl)OH. A particular value of
B is CH2OH.

[0062] In certain embodiments, B is CH(OH)CH2OH.

[0063] It will be appreciated that certain compounds according to the
invention may contain one or more centers of asymmetry and may therefore
be prepared and isolated in a mixture of isomers such as a racemic
mixture, or in an enantiomerically pure form.

[0064] It will further be appreciated that the compounds of Formula I or
their salts may be isolated in the form of solvates, and accordingly that
any such solvate is included within the scope of the present invention.

[0065] The compounds of Formula I include pharmaceutically acceptable
salts thereof. In addition, the compounds of Formula I also include other
salts of such compounds which are not necessarily pharmaceutically
acceptable salts, and which may be useful as intermediates for preparing
and/or purifying compounds of Formula I and/or for separating enantiomers
of compounds of Formula I.

[0066] According to another aspect, the present invention provides a
process for the preparation of a compound of Formula I or a salt thereof
as defined herein which comprises:

[0067] (a) coupling a corresponding compound having the formula II

##STR00004##

[0068] wherein L1 represents a leaving atom or group, with a compound
having the formula HNR10R11 (wherein NR10R11
represents a 4-8 membered heterocyclic ring optionally having an
additional heteroatom selected from N and O and optionally substituted
with one or more R9 groups, using a palladium catalyst and a ligand
in the presence of a base; or

[0069] (b) reacting a compound of Formula III

##STR00005##

[0070] with a compound having the Formula IV

##STR00006##

[0071] in the presence of an organo hypervalent iodine reagent; or

[0072] (c) for a compound of Formula I where B is --ORa, reacting a
corresponding compound having the Formula V

##STR00007##

[0073] with a compound of the formula Ra-L2, wherein L2
represents a leaving atom or group, in the presence of a base;

[0074] (d) for a compound of Formula I wherein B is
--(CH2)NRbRc, reacting a corresponding compound having the
Formula VI

##STR00008##

[0075] with hydrazine; and

[0076] removing any protecting group or groups and, if desired, forming a
salt.

[0077] Referring to method (a), the leaving atom L1 may be, for
example, a halide such as Br or I. Alternatively, L1 may be a
leaving group, such as a hydrocarbylsulfonyloxy group, for example, a
triflate group, or an arylsulfonyloxy group or an alkylsulfonyloxy group,
such as a tosylate or a mesylate group. Suitable palladium catalysts
include Pd2(dba)3 and Pd(OAc)2. Suitable ligands include
rac-BINAP or DIPHOS. The base may be, for example, an alkali metal
carbonate or alkoxide, such as for example cesium carbonate or sodium
tert-butoxide. Convenient solvents include aprotic solvents such as
ethers (for example tetrahydrofuran or p-dioxane) or toluene. The
coupling of a compound of formula (II) with HNR10R11 can be
conveniently performed at a temperature between 0° C. and reflux,
and more particularly at reflux.

[0078] Referring to method (b), the organo hypervalent iodine reagent
refers to any hypervalent iodine reagent suitable for forming
heterocyclic rings. Examples include iodobenzene diacetate (IBD) and
[hydroxy(tosyloxy)iodo]benzene (HTIB), which can be prepared by treating
IBD with p-toluenesulfonic acid monohydrate in acetonitrile. Suitable
solvent systems when using IBD include methanolic potassium hydroxide.
Suitable solvent systems when using HTIB include neutral solvents, for
example acetonitrile or dioxane. The reaction can be performed at a
temperature ranging from 0 to 60° C.

[0079] Referring to method (c), the leaving atom L2 may be, for
example a halogen atom such as Br, Cl or I. Alternatively, L2 can be
a leaving group, for example an arylsulfonyloxy group or an
alkylsulfonyloxy group, such as a mesylate or a tosylate group. The base
may be, for example, an alkali metal hydride or carbonate, such as sodium
hydride, potassium hydride, sodium carbonate, potassium carbonate or
cesium carbonate. Convenient solvents include aprotic solvents such as
ethers (for example tetrahydrofuran or p-dioxane), DMF, or acetone. The
reaction can be conveniently performed at a temperature ranging from -78
to 100° C.

[0080] Referring to method (d), the reaction is conveniently performed at
ambient temperature. Suitable solvents include alcohols such as methanol.

[0081] A compound of Formula II

##STR00009##

[0082] can be prepared by cyclizing a corresponding compound having the
formula VII

##STR00010##

[0083] where P is an alcohol protecting group, in the presence of an
organo hypervalent iodine reagent as described above, followed by
deprotection of the alcohol group and conversion of the alcohol group to
an alkylsulfonyloxy group, such as a triflate group.

[0084] The compounds of the formulas II and VII are believed to be novel
and are provided as further aspects of the invention.

[0085] The ability of test compounds to act as PIM-1, PIM-2 or PIM-3
inhibitors may be demonstrated by the assay described in Examples A, B
and C, respectively.

[0086] Compounds of Formula I have been found to be inhibitors of PIM-1
and/or PIM-2 and/or PIM-3, and are useful for treating diseases and
disorders which can be treated with a PIM-1 and/or PIM-2 and/or PIM-3
kinase inhibitor, including diseases mediated by PIM-1 and/or PIM-2
and/or PIM-3 kinases. Particular compounds of this invention are
inhibitors of PIM-1 and therefore are useful in treating diseases and
disorders mediated by PIM-1, such as cancers, such as hematological
cancers and solid tumors (e.g., breast cancer, colon cancer, gliomas).

[0088] In addition, certain compounds according to the present invention
may be useful for the treatment of inflammatory disorders mediated by T
and B cells function, such as rheumatoid arthritis, lupus, multiple
sclerosis, and inflammatory bowel disease.

[0089] Accordingly, a further embodiment of this invention provides a
method of treating cancer in a mammal in need thereof, comprising
administering to said mammal a compound of Formula I or a
pharmaceutically acceptable salt thereof. In one embodiment, the cancer
is of hematological origin. In one embodiment, the cancer derives from T
cells. In one embodiment, the cancer derives from B cells.

[0090] A further embodiment of this invention provides a compound of
Formula I or a pharmaceutically acceptable salt thereof, for use in the
treatment of cancer. In one embodiment, the cancer is of hematological
origin. In one embodiment, the cancer derives from T cells. In one
embodiment, the cancer derives from B cells.

[0091] Another embodiment of this invention provides a method of treating
or preventing inflammatory and autoimmune diseases, comprising
administering to a mammal in need thereof an effective amount of a
compound of Formula I. Examples of diseases which can be treated include
inflammatory or autoimmune diseases. In one embodiment, the disease is
multiple sclerosis. In another embodiment, the disease is lupus. In
another embodiment, the disease is inflammatory bowel disease.

[0092] A further embodiment of this invention provides a compound of
Formula I for use in treating an inflammatory or autoimmune disease. In
one embodiment, the disease is multiple sclerosis. In another embodiment,
the disease is lupus. In another embodiment, the disease is inflammatory
bowel disease.

[0093] Expression of PIM kinases in immune cells can be induced by
cytokines present during immune responses. Immune cells are critically
dependent on cytokines for differentiation and development of effector
functions during normal and pathogenic immune responses. Thus, compounds
of the invention may be useful for treating diseases and disorders
characterized by aberrant cytokine production and responses and/or
aberrant immune cell activation.

[0094] Accordingly, another embodiment of the invention provides a method
of treating diseases and disorders characterized by aberrant cytokine
production and responses and/or aberrant immune cell activation in a
mammal in need thereof, comprising administering to the mammal a compound
of Formula I or a pharmaceutically acceptable salt thereof.

[0096] Particular examples of diseases and disorders which can be treated
using a compound of Formula I include autoimmune and inflammatory
diseases. Particular examples of such diseases include asthma, MS,
inflammatory bowel disease (IBD), lupus, psoriasis and rheumatoid
arthritis.

[0097] Another embodiment provides a compound of Formula I or a
pharmaceutically acceptable salt thereof for use in the treatment of
diseases and disorders characterized by aberrant cytokine production and
responses and/or aberrant immune cell activation in a mammal. Examples of
such diseases and disorders include autoimmune and inflammatory diseases.

[0098] A subset of the triazolopyridine compounds disclosed herein was
found to have an IC50 for PIM-1 that is at least 10 fold less than
the IC50 for PIM-2. As a further example, particular
triazolopyridine compounds disclosed herein were found to have an
IC50 for PIM--that is at least 100 fold less than the IC50 for
PIM-2. Accordingly, also provided are triazolopyridine compounds which
are highly potent PIM-1 inhibitors and are highly selective for PIM-1
relative to PIM-2.

[0099] A subset of the triazolopyridine compounds disclosed herein were
found to have an IC50 for PIM-1 that is at least 10 fold less than
the IC50 for PIM-2 and an IC50 for PIM-3 approximately
equivalent to that observed for PIM-1. As a further example, particular
triazolopyridine compounds disclosed herein were found to have an
IC50 for PIM-1 that is at least 100 fold less than the IC50 for
PIM-2, and IC50 for PIM-3 approximately equivalent to that observed
for PIM-1. Accordingly, also provided are triazolopyridine compounds
which are highly potent PIM-1/PIM-3 dual inhibitors and are highly
selective for PIM-1 and PIM-3 relative to PIM-2.

[0100] As used herein, the term treatment includes prophylaxis as well as
treatment of an existing condition.

[0101] Accordingly, another aspect of this invention provides a method of
treating diseases or medical conditions in a mammal mediated by a PIM-1
and/or PIM-2 and/or PIM-3 kinase, comprising administering to said mammal
one or more compounds of Formula I or a pharmaceutically acceptable salt
or prodrug thereof in an amount effective to treat or prevent said
disorder.

[0102] Compounds of Formula I may also be useful as adjuvants to cancer
treatment, that is, they can be used in combination with one or more
additional drugs, for example a chemotherapeutic that works by the same
or by a different mechanism of action.

[0103] Compounds of the present invention may also be used in combination
with one or more additional drugs, for example an anti-inflammatory
compound, an immunosuppressive compound or an immunodepleting agent that
works by the same or a different mechanism of action.

[0104] The phrase "effective amount" means an amount of compound that,
when administered to a mammal in need of such treatment, is sufficient to
(i) treat or prevent a particular disease, condition, or disorder
mediated by a PIM-1 and/or PIM-2 and/or PIM-3 kinase, (ii) attenuate,
ameliorate, or eliminate one or more symptoms of the particular disease,
condition, or disorder, or (iii) prevent or delay the onset of one or
more symptoms of the particular disease, condition, or disorder described
herein.

[0105] The amount of a compound of Formula I that will correspond to such
an amount will vary depending upon factors such as the particular
compound, disease condition and its severity, the identity (e.g., weight)
of the mammal in need of treatment, but can nevertheless be routinely
determined by one skilled in the art.

[0106] As used herein, the term "mammal" refers to a warm-blooded animal
that has or is at risk of developing a disease described herein and
includes, but is not limited to, guinea pigs, dogs, cats, rats, mice,
hamsters, and primates, including humans.

[0107] Compounds of the invention may be administered by any convenient
route, e.g. into the gastrointestinal tract (e.g. rectally or orally),
the nose, lungs, musculature or vasculature, or transdermally or
dermally. Compounds may be administered in any convenient administrative
form, e.g. tablets, powders, capsules, solutions, dispersions,
suspensions, syrups, sprays, suppositories, gels, emulsions, patches etc.
Such compositions may contain components conventional in pharmaceutical
preparations, e.g. diluents, carriers, pH modifiers, sweeteners, bulking
agents, and further active agents. If parenteral administration is
desired, the compositions will be sterile and in a solution or suspension
form suitable for injection or infusion. Such compositions form a further
aspect of the invention.

[0108] According to another aspect, the present invention provides a
pharmaceutical composition, which comprises a compound of Formula I or a
pharmaceutically acceptable salt thereof, as defined hereinabove. In one
embodiment, the pharmaceutical composition includes the compound of
Formula I together with a pharmaceutically acceptable diluent or carrier.

[0109] According to another aspect, the present invention provides a
compound of Formula I or a pharmaceutically acceptable salt thereof, for
use in therapy, such as the treatment of a PIM-1 and/or PIM-2 and/or
PIM-3 kinase-mediated condition.

[0110] According to a further aspect, the present invention provides the
use of a compound of Formula I, or a pharmaceutically acceptable salt
thereof, for use in the treatment of a PIM-1 and/or PIM-2 and/or PIM-3
kinase-mediated condition, as defined hereinabove.

EXAMPLES

[0111] The following examples illustrate the invention. In the examples
described below, unless otherwise indicated all temperatures are set
forth in degrees Celsius. Reagents were purchased from commercial
suppliers such as Aldrich Chemical Company, Lancaster, TCI or Maybridge,
and were used without further purification unless otherwise indicated.
Tetrahydrofuran (THF), dichloromethane (DCM, methylene chloride),
toluene, and dioxane were purchased from Aldrich in Sure seal bottles and
used as received.

[0112] The reactions set forth below were done generally under a positive
pressure of nitrogen or argon or with a drying tube (unless otherwise
stated) in anhydrous solvents, and the reaction flasks were typically
fitted with rubber septa for the introduction of substrates and reagents
via syringe. Glassware was oven dried and/or heat dried.

[0113] Column chromatography was done on a Biotage system (Manufacturer:
Dyax Corporation) having a silica gel column or on a silica SepPak
cartridge (Waters).

Example A

Enzyme PIM-1 Assay

[0114] The assay for the determination of PIM-1 activity is based on the
incorporation of [33P]PO4 from [γ-33P]ATP into
PIM2tide substrate and capture of the radiolabeled peptide onto a Whatman
P81 (phosphocellulose) filter plate. The amount of radiolabeled product
is then measured by liquid scintillation counting. The final buffer
conditions were as follows: 20 mM K+MOPS, pH 7.4, 10 mM MgCl2,
0.005% Tween-20, 1 mM DTT. Assay mixtures contained 35 μM
[γ-33P]ATP (20 μCi/mL), 7.5 μM PIM2tide and 0.25 nM
PIM-1 in a total volume of 50 μL. Incubations were carried out for 60
min at 22° C. and quenched with 75 μL, of 200 mM
H3PO4, filtered through a Whatman P81 plate and washed
(1×200 μL and 5×100 μL) with 200 mM H3PO4.
Fifty μL of liquid scintillation cocktail were then added per well,
and the plate was counted for 30 s/well using a TopCount NXT.

[0115] IC50 Determinations:

[0116] Compounds were prepared at 50× the final concentration in
DMSO by conducting 3-fold serial dilutions from a 500-μM intermediate
dilution to give a 10-point dosing curve having a high dose of 10 μM.
One-μL aliquots of these were then transferred to the assay mixtures
above to give a final concentration of DMSO of 2%. A standard or
reference compound was typically included on each assay plate to validate
that plate. For each plate, percent of control (POC) values were
calculated for each well. IC50's were estimated from the POC's using
a standard 4-parameter logistic model. The IC50 is defined as the
concentration of inhibitor at which the POC equals 50 for the fitted
curve. Compounds of Formula I were found to have an average IC50
below 10 μM when tested in this assay. Specific IC50 values are
provided in Table 1.

Example B

PIM-2 Assay

[0117] Assay was performed as described in Example A, using 4 μM
[γ-33P]ATP (20 μCi/mL), 1.0 μM PIM2tide and 1.5 nM
GST-tagged recombinant full-length human Pim-2 in place of PIM-1.
Compounds of Formula I were found to have an average IC50 below 10
μM when tested in this assay. Specific IC50 values are provided
in Table 1.

Example C

PIM-3 Assay

[0118] Assay was performed as described in Example A, using 30 μM
[γ-33P]ATP (20 μCi/mL), 3.75 μM PIM2tide and 0.5 nM
recombinant rat PIM-3 in place of PIM-1. Compounds of Formula I were
found to have an average IC50 below 10 μM when tested in this
assay. Specific IC50 values are provided in Table 1.

[0119] The assay for determination of the antiproliferative activity of
multiple PIM inhibitors in the JAK2-driven cell lines is conducted as
follows. Cells are plated out to 96-well plates at an initial density of
10,000 cells/well in 95 μL. Compounds are prepared at 20× the
final concentration in DMSO by conducting 3-fold serial dilutions to give
a 10-point dosing curve having a high dose of 1000 μM. Aliquots (5
pit) of these dilutions are then transferred to the appropriate wells of
the 96-well plates containing cells to yield a final DMSO concentration
of 0.5%. The cells are then incubated with compound for 72 hours at
37° C., 5% CO2. CelltiterBlue reagent (Promega, Catalog #:
G8080) is then added (20 μL/well) and incubated at 37° C., 5%
CO2 for 1-8 hours depending on the cell line being analyzed. The
plate is then quantified employing a fluorescence plate reader (Model:
Gemini [Molecular Devices]; Settings: 560 nm (Ex)/590 nm (Em) 570 nm
(cut-off) [CellTiter Blue Assay].

[0120] The values for each well are then converted to a percent of
untreated control (POC). These POC values are then plotted as a function
of compound concentration. A 4-parameter curve-fit analysis is performed
for each compound dilution and an IC50 value is calculated from this
curve.

Example E

T Cell In Vitro Functional Assays

[0121] The in vitro assays which can be used to assess the effects of the
compounds of the invention are described in assays A, B, C and D below.
CD4+ T cells are isolated from red blood cell-depleted splenocytes of
C57Bl/6J mice (Jackson Laboratories, catalog #000664) using CD4+ T cell
isolation kit (Miltenyi, catalog #130-090-860).

[0122] In assay (A), purified CD4+ T cells are plated in 96 well plates at
90000 cells/well in 90 μL. A dilution series of the compounds are
prepared at 100× the final concentration in DMSO and then diluted
10-fold into complete media (10× stocks). 10 pit of 10×
compound stocks are added to appropriate wells of 96 well plates
containing cells and incubated for 1 hour at 37° C., 5% CO2.
The cell/compound mixtures are then transferred to a 96 well plate coated
with anti-CD3 mAb (1 μg/mL; BD Pharmingen, catalog #553057) and
soluble anti-CD28 mAb (1 μg/mL; BD Pharmingen, catalog #553294) was
added. Plates are cultured at 37° C., 5% CO2 for 40 hours. 20
μL of the culture are removed for determination of proliferation using
the CellTitre-Glo® luminescent assay (Promega, Catalog #G7571)
according to the manufacturer's protocol. The plate is quantified on a
Packard TopCount instrument using luminescence protocol and data analyzed
using Prism software.

[0123] In assay (B), purified CD4+ cells are treated with compound and
stimulated as described for assay (A). After 40 hours, supernatants are
assayed for IL-2 using R&D duo set ELISA kits (catalog #DY402). ELISA
plates are quantified relative to a standard curve using Molecular
Devices Versamax Reader at 450 nM and Softmax Pro software.

[0124] In assay (C), 1,000,000 cells/mL of purified CD4+ T cells are mixed
with 1 μg/mL anti-CD28, 10 ng/mL IL-4 (R&D Systems cat #404-ML-010/CF)
and 2 μg/mL anti-IFNγ (R&D Systems catalog #AB-485-NA) and
placed into plates coated with 1 μg/mL anti-CD3. After 5 days, cells
are harvested, washed and incubated overnight at 37° C., 5%
CO2. The following day, 50,000 cells are plated into each well of a
96 well plate. A dilution series of compounds are prepared at 200×
the final concentration in DMSO, then 10× stocks are prepared by
dilution in cell culture media. 10 μL of 10× stocks are added to
the cells in the 96-well plate and incubated for 2 hours at 37°
C., 5% CO2. Cell/compound mixtures are then transferred to well
coated with 0.1 μg anti-CD3 and incubated at 37° C., 5%
CO2. Culture supernatants are removed 18 hours later and tested for
IL-4 levels by ELISA (R&D Systems catalog #DY404). ELISA plates are
quantified relative to a standard curve using Molecular Devices Versamax
Reader at 450 nM and Softmax Pro software.

[0126] The effect of compounds of Formula I on T cell responses can be
determined by the following experiment. On Day 0, C57BL/6 (Jackson
Laboratories #000664, 6-8 weeks of age) are immunized at the base of the
tail with 100 μg of hen egg lysozyme (HEL; Sigma #L7773) with complete
Freund's adjuvant (CFA; Sigma #F5881). Starting on Day 0 and continuing
until Day 7, mice are dosed twice a day by oral administration with
vehicle (water) or the compound of Formula I (200 mg/kg). On Day 7,
popiteal lymph nodes are removed, single cell suspensions are prepared
and 500,000 cells in 200 μL are activated in 96 well plates with the
indicated dose of HEL peptide. Following incubation for 72 hours at
37° C., 5% CO2, supernatants are harvested for IFNγ
ELISA (R&D Systems catalog #MIF00) and proliferation is assessed using
the CellTitre-Glo® luminescent assay (Promega, Catalog #G7571) with
both assays performed according to the manufacturer's protocol. ELISA
plates are quantified relative to a standard curve using Molecular
Devices Versamax Reader at 450 nM and Softmax Pro software; proliferation
was quantitated on a Packard TopCount instrument using luminescence
protocol and data analyzed using excel software.

Example G

B Cell In Vivo Functional Assay

[0127] The effect of a compound of Formula I on B cell responses can be
determined with the following experiment. On Day 0, C57BL/6J mice
(Jackson Laboratories #000664, 6-8 weeks of age) are immunized at the
base of the tail with 20 μg of hen egg lysozyme (HEL; Sigma #L7773)
with complete Freund's adjuvant (CFA; Sigma #F5881). Mice are
re-immunized on day 7 with 20 μg HEL in alum (Pierce catalog #77161).
Starting on Day 0 and continuing through Day 28, mice are dosed once a
day by oral administration with vehicle (water) or the compound of
Formula I (200 mg/kg). Serum is collected on days 0, 7, 14, 21, and 28
and analyzed for HEL-specific total IgG, IgG1, IgG2a, IgG2b, and IgG3
antibody production by capture ELISA (antibodies purchased from
Invitrogen, catalog Nos. M30007, M32107, M32307, M32507 and M32607).
ELISA plates are quantitated using Molecular Devices Versamax reader at
450 nM. The group mean titer of each antibody analyte is converted to
percent of vehicle control (=100%).

Example H

Adoptive Transfer Experimental Autoimmune Encephalomyelitis

[0128] The effect of a compound of Formula I on an autoimmune disease
induced by T cells can be determined using an adoptive transfer EAE
model, an animal model of human multiple sclerosis (Brain (2006), 129,
1953-1971). This model relies on the injection of T cells from animals
with EAE into disease-free host animals. This injection of cells is known
to those skilled in the art as adoptive transfer. By injecting the
animals with activated, encephalogenic T cells, this model is focused on
the pathogenic stage of EAE autoimmune disease. On Day -14, C57BL/6 mice
(Taconic Farms; 10 weeks old) are immunized with a disease-causing
protein, MOG(35-55) peptide in complete Freund's adjuvant (Hooke
Laboratories, catalog #EK-0113). On Day -3, spleens are harvested, single
cell suspensions are prepared and then 5,000,000 cells/mL are stimulated
with 20 μg/mL MOG(33-55) peptide (Open Biosystems), 30 ng/mL IL-12
(R&D Systems catalog #419-ML-010), 10 μg/mL anti-IFNγ antibody
(BD Biosciences catalog #554408) at 37° C., 5% CO2. On Day 0,
1,500,000 of these cells are injected intravenously into the tail veins
of C57BL/6 recipient mice. The recipient mice are divided into treatment
groups for vehicle (distilled water; 10 mL/kg) or the compound of Formula
I (200 mg/kg), both administered by oral gavage twice daily for 26 days.
The recipient mice are scored daily days 0 through 26 using the following
clinical scoring system:

[0129] 0.0--no symptoms

[0130] 1.0--limp tail

[0131] 2.0--limp tail and weakness of hind legs

[0132] 3.0--limp tail and complete hind limb paralysis, or partial front
and hind limb paralysis, or severe head tilting combined with pushing
against cage wall and spinning when picked up by tail

[0147] The following adoptive transfer model of inflammatory bowel disease
(IBD) can be performed to determine the effect of compounds of Formula I
on IBD, which is an autoimmune disease associated with T cells and
cytokines.

[0148] On Day 0, CD4+ T cells are isolated from the spleens of female
Balb/cAnNCrl mice (Charles River Laboratories; 12 weeks old) as described
in Example E. The resulting cells are labeled with fluorescent antibodies
against CD4 and CD45 markers and are sorted by flow cytometry for
CD4+CD45RBhi cells based on fluorescence. 400,000 CD4+CD45RBhi cells are
then injected intraperitoneally into C.B17/Icr-Prkdescid/IcrIcoCrl
mice (Charles River Laboratories strain code 236; 12 weeks old). This
injection of cells is known to those skilled in the art as "adoptive
transfer". On Day 21, mice are randomized into groups for oral gavage
treatment with vehicle (1% carboxymethylcellulose sodium (CMC)/0.5% Tween
80 once daily; CMC, Sigma catalog #C9481, Tween 80 Sigma catalog #P1754)
or the compound of Formula I (200 mg/kg; twice daily). Treatments
continued through Day 42.

[0149] At the conclusion of the study, mice are sacrificed and the distal
half of their colons are placed in 10% neutral buffered formalin (Richard
Allen Scientific catalog #53120-1) and paraffin embedded, sectioned into
4 μm slices and stained with hematoxylin and eosin (H&E) for analysis
by a board certified veterinary pathologist.

[0150] For each H&E stained section, submucosal edema is quantitated by
measuring the distance from the muscularis mucosa to the internal border
of the outer muscle layer in a non-tangential area thought to most
representative the severity of this change. Mucosal thickness is also
measured in a non-tangential area of the section that best represented
the overall mucosal thickness. This parameter is indicative of gland
elongation and mucosal hyperplasia. The extent of inflammation
(macrophage, lymphocyte and polymorphonuclear leukocyte (PMN) infiltrate)
is assigned severity scores according to the following criteria:

[0151] Normal=0

[0152] Minimal=1 (generally focal affecting 1-10% of mucosa or if diffuse
then minimal)

[0153] Mild=2 (generally focal affecting 11-25% of mucosa or if diffuse
then mild)

[0164] Parameters that are scored using % involvement included: colon
glandular epithelial loss (this includes crypt epithelial as well as
remaining gland epithelial loss), and colon erosion (this reflects loss
of surface epithelium and generally is associated with mucosal hemorrhage
(reflective of the bleeding seen clinically and at necropsy).

[0165] The three scored parameters (inflammation, glandular epithelial
loss, and erosion) are ultimately summed to arrive at a sum of
histopathology scores, which indicates the overall damage and would have
a maximum score of 15.

Example K

MRL/lpr Lupus Model

[0166] MRL/lpr is considered to be an animal model of systemic lupus
erythematosus (SLE), an autoimmune disease (Cohen and Maldonado 2003,
Current Protocols in Immunology Chapter 15, Unit 15.20). MRL/lpr mice
have a defect in the apoptosis of activated lymphocytes and over time
develop a spontaneous and severe lymphoproliferative disorder
characterized by enlarged lymphoid organs, auto-antibody production and
kidney disease resulting in proteinuria. SLE patients also exhibit
auto-antibodies, and some patients develop kidney disease. To determine
the effect of compounds of Formula I in this model of SLE, the following
experiment can be conducted.

[0167] MRL/MpJ-Fas<lpr> and age-matched MRL/MpJ control mice
(Jackson Laboratories, catalog #000485 and #000486, respectively) are
treated once daily with vehicle (1% CMC/0.5% Tween 80) or twice daily
with the compound of Formula I (200 mg/kg) for 10 weeks. Body weights,
lymphadenopathy and urine protein levels are monitored weekly. Urine
protein levels are determined with Bayer Albustix dipsticks (Bayer
catalog #2191) and scored according to the following scale:

[0168] 0=none detected

[0169] 0.5=trace amounts

[0170] 1=30 mg/dL

[0171] 2=100 mg/dL

[0172] 3=300 mg/dL

[0173] 4=2000 mg/dL

[0174] Serum levels of anti-ds-DNA antibody are measured by ELISA (Alpha
Diagnostic, catalog #5120) on Day 28 and upon study termination. ELISA
plates are quantitated using a Molecular Devices Versamax plate reader at
450 nM and titers calculated relative using to a standard curve using a
4-parameter curve fit with Softmax Pro software.

[0176] Potassium 2-methylpropan-2-olate (4.214 g, 35.678 mmol) was slowly
added under an atmosphere of dry N2 to a solution of
2-chloro-4-nitropyridine (5.142 g, 32.434 mmol) in 2-methoxyethanol (40.0
mL, 506.74 mmol). The reaction was stirred at ambient temperature for 2
hours and then concentrated under reduced pressure. The resulting oil was
diluted with water (200 mL) and extracted with EtOAc. The combined
organic phases were dried over Na2SO4, filtered and
concentrated under reduced pressure to give 5.36 g (88%) of desired
product as a colorless oil.

Step B: Preparation of 2-hydrazinyl-4-(2-methoxyethoxy)pyridine

[0177] Hydrazine (10 mL, 318.6 mmol) was added to a solution of
2-chloro-4-(2-methoxyethoxy)pyridine (1.00 g, 5.330 mmol) in pyridine (25
mL). The reaction was heated to reflux. After 18 hours the reaction
mixture was partitioned between H2O and DCM and the aqueous phase
was extracted with DCM. The combined organic extracts were dried over
Na2SO4, filtered and concentrated under reduced pressure. The
residue was purified via flash column chromatography (40:1 DCM/MeOH
followed by 20:1 DCM/MeOH) to provide 320 mg (33%) of desired product as
a white solid.

[0178] 8-Hydroxyquinoline-2-carbaldehyde (5.00 g, 28.9 mmol) and imidazole
(4.32 g, 63.5 mmol) were dissolved in DCM (50 mL) under N2. The
reaction mixture was cooled to 0° C. after which time
tert-butylchlorodimethylsilane (4.94 g, 31.8 mmol) was added. After
stirring for 16 hours at ambient temperature, the reaction mixture was
partitioned between DCM and H2O. The organic layer was washed with
H2O and aqueous saturated NaHCO3, dried over Na2SO4,
filtered and concentrated under reduced pressure to afford an orange oil.
The residue was purified via flash column chromatography (10:1
Hexane/EtOAc) to provide 6.85 g (83%) of desired product as a
yellow/orange oil.

[0181] To a solution of
8-(tert-butyldimethylsilyloxy)-2-(7-(2-methoxyethoxy)-[1,2,4]triazolo[4,3-
-a]pyridin-3-yl)quinoline (0.070 g, 0.155 mmol) in THF (8 mL) was added 1M
HCl (1.5 mL, 1.50 mmol). After stirring at ambient temperature for 2
hours 1 M HCl (5 mL) was added and the reaction mixture stirred for a
further 16 hours. The mixture was neutralized with 1M NaOH and diluted
with EtOAc. The organic layer was washed with brine, dried over
Na2SO4, filtered and concentrated under reduced pressure to
give 40 mg (77%) of desired product.

[0188] Prepared as described in Example 1 using 2-chloro-4-methoxypyridine
in place of 2-chloro-4-(2-methoxyethoxy)pyridine in step B, and
substituting tert-butyl piperidin-4-ylcarbamate for tert-butyl
piperazine-1-carboxylate in step H. MS APCI (+) m/z 375.2 (M+1) detected.

[0190] Prepared as described in Example 1 using 2-chloro-4-methoxypyridine
in place of 2-chloro-4-(2-methoxyethoxy)pyridine in step B, and
substituting morpholine for tert-butyl piperazine-1-carboxylate in step
H. MS APCI (+) m/z 362.3 (M+1) detected.

[0192] Prepared as described in Example 1 using 2-chloropyridine as a
replacement for 2-chloro-4-(2-methoxyethoxy)pyridine in step B, and
substituting piperidine for tert-butyl piperazine-1-carboxylate in step
H. MS ESI (+) m/z 330 (M+1) detected.

[0196] Prepared as described in Example 1 using 2-chloropyridine as a
replacement for 2-chloro-4-(2-methoxyethoxy)pyridine in step B, and
substituting (R)-tert-butyl 2-methylpiperazine-1-carboxylate for
tert-butyl piperazine-1-carboxylate in step H. MS ESI (+) m/z 345 (M+1)
detected.

[0198] Prepared as described in Example 1 using 2-chloropyridine as a
replacement for 2-chloro-4-(2-methoxyethoxy)pyridine in step B, and
substituting (cis)-tert-butyl 2,6-dimethylpiperazine-1-carboxylate for
tert-butyl piperazine-1-carboxylate in step H. MS ESI (+) m/z 359 (M+1)
detected.

[0200] Prepared as described in Example 1 using 2-chloropyridine as a
replacement for 2-chloro-4-(2-methoxyethoxy)pyridine in step B, and
substituting tert-butyl piperidin-4-ylcarbamate for tert-butyl
piperazine-1-carboxylate in step H. MS ESI (+) m/z 345 (M+1) detected.

[0202] Prepared as described in Example 1 using 2-chloropyridine as a
replacement for 2-chloro-4-(2-methoxyethoxy)pyridine in step B, and
substituting tert-butyl piperidin-4-ylmethylcarbamate for tert-butyl
piperazine-1-carboxylate in step H. MS ESI (+) m/z 359 (M+1) detected.

[0209] Sodium periodate (0.34 mL, 0.22 mmol was added drop-wise to a
slurry of silica gel (0.4 g) in DCM (3 mL)). The reaction was stirred for
10 minutes. tert-Butyl
(1-(2-(1,2-dihydroxyethyl)quinolin-8-yl)piperidin-4-yl)methylcarbamate
(74 mg, 0.18 mmol) in DCM (2 mL) was then added to the slurry and the
reaction was stirred for 30 minutes. The reaction was filtered and the
collected solids were washed with DCM (20 mL). The filtrate was
concentrated to give the crude product.

[0210] To tert-butyl
(1-(2-formylquinolin-8-yl)piperidin-4-yl)methylcarbamate (112 mg, 0.303
mmol) in DCM (20 mL) was added 1-(4-iodopyridin-2-yl)hydrazine (78.4 mg,
0.333 mmol). The reaction mixture was stirred for 1 hour and then taken
on to directly to the next step.

[0211] To (E)-tert-butyl
(1-(2-((2-(4-iodopyridin-2-yl)hydrazono)methyl)quinolin-8-yl)piperidin-4--
yl)methylcarbamate (178 mg, 0.304 mmol) was added iodobenzene diacetate
(IBD) (127 mg, 0.395 mmol). The reaction was stirred for 2 hours.
Additional IBD (0.5 eq) was added and the reaction was stirred for
another 2 hours and then quenched with saturated Na2S2O3
(5 mL). The organic layer was extracted with DCM, dried
(Na2SO4) and concentrated. The crude material was purified by
silica gel chromatography (EtOAc/Hexane/MeOH 2:1:0.1) to provide the
final product.

[0224] Prepared as described in Example 1, steps D-E, using tert-butyl
(1-(2-formylquinolin-8-yl)piperidin-4-yl)methylcarbamate in place of
8-(tert-butyldimethylsilyloxy)quinoline-2-carbaldehyde, and
2-hydrazinyl-4-iodopyridine in place of
2-Hydrazinyl-4-(2-methoxyethoxy)pyridine.

[0229] Prepared as described in Example 1 using 2-chloropyridine as a
replacement for 2-chloro-4-(2-methoxyethoxy)pyridine in step B, and
piperidin-3-ylmethanol for tert-butyl piperazine-1-carboxylate in step H.
MS ESI (+) m/z 360.3 (M+1) detected.

[0231] 2-Bromo-4-fluorobenzenamine (10 g, 52.6 mmol) was weighed into a
round bottom flask, and dissolved in 40 mL of 6N HCl. The reaction
mixture was then heated to reflux, followed by drop-wise addition of
(E)-but-2-enal (4.578 mL, 55.3 mmol) mixed with 1.0 mL deionized water
over 25 minutes. Following complete addition the reaction was heated at
100° C. for an additional 35 minutes, until all the
2-bromo-4-fluorobenzenamine had been consumed. The reaction was cooled to
ambient temperature, followed by addition of 50 mL of Et2O. The
reaction was stirred for 5 minutes followed by removal of Et2O by
partitioning. The aqueous layer was replaced into the original reaction
flask and ZnCl2 (3.586 g, 26.3 mmol) was then added in two portions
followed by cooling to 0° C. over 30 minutes. The pH of the crude
reaction mixture was then adjusted to pH=8.0 using concentrated
NH4OH. The crude mixture was then extracted with Et2O, followed
by ethyl acetate. The combined organics were then dried over
Na2SO4, and then concentrated in vacuo, affording the desired
product as a brown solid (10.7 g, 85% yield). MS APCI (+) m/z 240.2 and
242.2 (M+1 of each Br isotope) detected.

Step B: Preparation of 8-bromo-2-(dibromomethyl)-6-fluoroquinoline

[0232] 8-Bromo-6-fluoro-2-methylquinoline (10.7 g, 44.6 mmol) was weighed
into a 1 neck flask, followed by addition of NaOAc (21.9 g, 267 mmol).
The solids were suspended in 500 mL of AcOH, and the reaction heated to
70° C. Bromine (6.85 mL, 134 mmol) was the added drop-wise over 25
minutes as a solution in 30 mL of AcOH. Following complete addition, the
reaction was stirred at 100° C. for 1 hour. The reaction was then
cooled to ambient temperature, followed by pouring onto 750 cc of ice.
The ice was allowed to melt completely and the brown slurry separated by
partitioning 4×400 mL ethyl acetate. The combined organics were
then dried over MgSO4, and concentrated in vacuo to afford a brown
solid (17.2 g, 97% yield).

[0233] 8-Bromo-2-(dibromomethyl)-6-fluoroquinoline (17.2 g, 43.2 mmol) was
weighed into a flask and dissolved in 250 mL of EtOH, followed by
addition of silver nitrate (23.5 g, 138 mmol) in 100 mL of 1:1
EtOH/H2O. The reaction was heated to reflux for 1 hour, at which
time all starting material had been consumed. The reaction was removed
from heat and filtered hot through a medium frit scintered glass funnel,
affording 5.84 g of 8-bromo-6-fluoroquinoline-2-carboxylic acid as a
white/yellow powder. The mother liquor was concentrated in vacuo,
followed by extractive work-up (200 mL ethyl acetate/water). The combined
organics were dried over Na2SO4 and concentrated in vacuo to
afford the desired products as an orange-brown semi solid (99% overall;
6.4 g and 5.8 g respectively). MS APCI (+) m/z 298 and 300 (M+1 of each
isotope) detected; MS APCI (-) m/z 268 and 269.9 (M-1 of each Br isotope)
detected.

Step D: Preparation of (8-bromo-6-fluoroquinolin-2-yl)methanol

[0234] Ethyl 8-bromo-6-fluoroquinoline-2-carboxylate (3.201 g, 10.7 mmol)
was weighed into a flask, and dissolved in 100 mL of DCM. The reaction
was cooled to -78° C., followed by drop-wise addition of DIBAL-H
(21.48 mL, 32.22 mmol) over 10 minutes. The reaction was then allowed to
stir and warm to ambient temperature over 2 hours at which time the
starting material had been consumed. The reaction was quenched with 10 mL
MeOH, followed by addition of 100 mL of Rochelle's Salts, and stirred
overnight to remove the emulsion. The reaction was then partitioned with
ethyl acetate. The combined organic fractions were concentrated in vacuo.
The crude semi solid was purified by flash column chromatography (eluting
with a 20-50% ethyl acetate/hexanes gradient), affording the desired
product as an orange-yellow semi solid (2.27 g, 42% yield) MS APCI (+)
m/z 256.1 and 258 (M+1 of each Br isotope) detected.

Step E: Preparation of 8-bromo-6-fluoroquinoline-2-carbaldehyde

[0235] (8-bromo-6-fluoroquinolin-2-yl)methanol (2 g, 7.8 mmol), DMSO (8.9
mL, 125.0 mmol), and TEA (4.9 mL, 35 mmol) were weighed into a flask and
dissolved in a 10 mL of DCM, followed by cooling to 0° C.
Pyridinium sulfate (4.351 g, 27.3 mmol) was added and the reaction
stirred 0° C. for 1 hour. The reaction was poured onto 50 mL water
and extracted with ethyl acetate. The combined organics were then dried
over MgSO4, then concentrated in vacuo affording a yellow/white
semi-solid, which was further purified by triturating with 20% ethyl
acetate/Hexanes, affording the desired product as a tan solid (1.35 g,
68% yield).

[0236] 8-Bromo-6-fluoroquinoline-2-carbaldehyde (100 mg, 0.39 mmol) and
1-(pyridin-2-yl)hydrazine (43 mg, 0.39 mmol) were dissolved in 15 mL of
absolute EtOH, and heated to reflux for 2 hours, at which time all
starting material had been consumed. The reaction was then cooled to
ambient temperature, and the yellow/orange product was collected by
filtration (wash EtOH), affording (100 mg, 73.6% yield) as a yellow
solid. MS APCI (+) m/z 345.1 and 347 (M+1 of each Br isotope) detected.

[0237] (E)-1-((8-Bromo-6-fluoroquinolin-2-yl)methylene)-2-(pyridin-2-yl)hy-
drazine (100 mg, 0.290 mmol) was weighed into a 25 mL 1 neck round bottom
flask and suspended in 6.0 mL of DCM, followed by addition of iodobenzene
diacetate (103 mg, 0.32 mmol). The reaction was stirred at 23° C.
overnight, after which all starting materials had been consumed. The
reaction was transferred to a separatory funnel and partitioned between
30 mL DCM and 30 mL Na2SO3, and the aqueous layer was washed
with DCM. The combined organics were dried over magnesium sulfate and
concentrated in vacuo to afford a white/yellow powder. The powder was
triturated with 10 mL of anhydrous Et2O, affording the desired
product as a white solid (74 mg, 74.4% yield). MS APCI (+) m/z 343 (M+1)
detected.

[0250] (R)-tert-butyl
1-(2-([1,2,4]triazolo[4,3-a]pyridin-3-yl)quinolin-8-yl)piperidin-3-ylcarb-
amate (0.030 g, 0.067 mmol) was added to a 1:1 TFA-DCM and stirred for
about 2 hours, at which time the reaction appeared complete by LC/TLC.
The crude mixture was evaporated and purified by flash column
chromatography (Horizon, using a gradient elution of 5% NH4 in
MeOH/DCM). The desired product was isolated as a light yellow solid (10
mg; 43% yield). MS ESI (+) m/z 345.2 (M+1) detected.

[0258] To a 250 mL flask was added toluene (150 mL) and t-BuNH2 (7.26
mL, 69.1 mmol). The solution was cooled to -25° C. and bromine
(1.95 mL, 38.0 mmol) was added. The solution was cooled to -78° C.
and 2-methylquinolin-8-ol (5.5 g, 34.6 mmol) was added as a
CH2Cl2 solution (15 mL). The reaction mixture was then
gradually warmed to ambient temperature over 6 hours. The mixture was
washed with water (50 mL) and then treated with 3.0 M aqueous NaOH (250
mL). This provided copious amounts of precipitate, which went into
solution after about 600 mL water was added. The layers were mixed and
separated. The alkaline extract was carefully acidified with concentrated
HCl (about 50 mL). The solution was extracted with CH2Cl2
(4×200 mL), and the combined extracts were washed with brine and
dried over Na2SO4, filtered and concentrated. The original
water wash was found to contain a significant amount of product, so 10 mL
1M HCl was added and the acidic solution was extracted with
CH2Cl2 (2×75 mL) and these layers were also washed with
brine, dried over Na2SO4, filtered and concentrated. The
organic phases were combined to provide 5.0 g (60%) of the desired
product as a red/brown solid.

[0259] To the product from Step A (3.0 g, 12.6 mmol) was added imidazole
(1.89 g, 27.7 mmol) and CH2Cl2 (40 mL). The solution was cooled
to 0° C. and then tent-butylchlorodimethylsilane (2.09 g, 13.9
mmol) was added in one portion. The reaction was gradually warmed to
ambient temperature over 1 hour and then stirred overnight. The mixture
was diluted with a saturated aqueous NH4Cl solution (25 mL) and
CH2Cl2 (40 mL). The layers were mixed and separated and the
organic layer was washed with brine and dried over Na2SO4,
filtered and concentrated. The crude product was purified by column
chromatography (2 to 20% CH2Cl2/hexanes) to provide 3.36 g
(76%) of the desired product as a white solid.

[0260] A slurry of SeO2 (0.869 g, 7.83 mmol) and 1,4-dioxane (20 mL)
was warmed to 80° C. and then the product from Step B (2.3 g, 6.53
mmol) was added as a 1,4-dioxane solution (20 mL). The mixture was
stirred at 80° C. for 32 hours. The mixture was cooled to ambient
temperature and filtered through GF/F filter paper and the residual solid
was washed with CH2Cl2. The filtrate was concentrated and
purified by passing through a silica gel plug, eluting with 50%
CH2Cl2/hexane to provide 2.14 g (89%) of the product as a
yellow/orange solid.

[0261] To the product from Step C (2.95 g, 8.05 mmol) was added EtOH (30
mL, anhydrous). To this solution was added 2-hydrazinylpyridine (0.967 g,
8.86 mmol). The mixture was stirred at ambient temperature for 24 hours.
The resulting precipitate was isolated by vacuum filtration and washed
with cold EtOH and then dried in vacuo to afford 2.98 g (73%) of the
desired product.

[0263] To the product from Step E (2.8 g, 6.15 mmol) was added THF (60
mL). The solution was cooled to 0° C. then TBAF.3H2O (2.33 g,
7.38 mmol) was added and the mixture was stirred for 1 hour. The mixture
was then diluted with EtOAc (100 mL) and then washed with saturated
aqueous NaHCO3 (75 mL). The layers were separated and the aqueous
phase washed with EtOAc (100 mL). The combined organic phases were washed
with brine and dried over Na2SO4, filtered and concentrated.
The crude mixture was triturated with MeOH and the mixture was filtered
and the solid was washed with Et2O (0.730 g; 35%).

[0264] The product from Step F (0.730 g, 2.14 mmol) was suspended in THF
(6 mL) and DMF (2 mL). NEt3 (0.746 mL, 5.35 mmol) was added followed
by N-Phenyltriflimide (0.917 g, 2.57 mmol). The mixture was stirred at
ambient temperature overnight. There was still starting material present,
so additional N-Phenyltriflimide (0.300 g) was added and the reaction
stirred for an additional 1.0 hours. The mixture was diluted with water
(25 mL), stirred for 30 min and then filtered. The solids were washed
with water, Et2O (10 mL) and then hexanes. The solid was dried in
vacuo until constant weight 0.890 g (88%) and was used directly in the
next step.

[0265] The product from Step G (0.025 g, 0.0528 mmol) and
3-methylpiperidine (0.0186 mL, 0.158 mmol) were added to a small
microwave reaction vial followed by NMP (0.250 mL). The vessel was placed
inside the center of the microwave oven and then it was exposed to
microwave irradiation (250 W) for 20 minutes at a temperature of
195° C. After the irradiation, the reaction mixture was cooled to
ambient temperature, poured into 10 mL of water and extracted with
CH2Cl2 (3×10 mL). The combined organic extracts were
washed with saturated brine and dried over anhydrous Na2SO4.
After removal of the solvent, the residue was purified by column
chromatography on silica gel to give the product as a dark orange
semisolid which was 80% pure. This material was purified via Preparative
TLC using 50% Acetone/hexane as eluent to provide 0.005 g (22%) of the
product as an orange film. MS ESI (+) m/z 422.3 (M) detected.

[0268] To the product from Step A (0.115 g, 0.233 mmol) was added HCl
(1.17 mL, 7.00 mmol, 6.0N aqueous). The mixture was stirred at 90°
C. for 45 minutes. The mixture was then cooled to ambient temperature and
diluted with water (10 mL) and with CH2Cl2 (10 mL). The layers
were mixed and separated and the aqueous phase was washed once more with
CH2Cl2 (10 mL) and was then treated with saturated aqueous
Na2CO3 until pH=10. The aqueous phase was then extracted with
CH2Cl2 (3×10 mL). The combined organic phases were washed
with water followed by brine and then dried over Na2SO4,
filtered and concentrated to provide 0.058 g (66%) of the product >96%
pure by HPLC. MS ESI (+) m/z 359.1 (M+1) detected.

[0270] To a 1.0 L flask was added tert-butyl 4-oxopiperidine-1-carboxylate
(150.0 g, 752.8 mmol), which was dissolved in DMF (400 mL). To this
solution was added TMS-Cl (114.7 mL, 903.4 mmol), followed by NEt3
(251.8 mL, 1807 mmol). The resulting heterogeneous mixture was warmed to
70° C. and stirred overnight under a N2 atmosphere. The
mixture was cooled to ambient temperature, diluted with hexanes (250 mL)
and filtered. The solids were washed with hexanes (4×250 mL). The
combined organic phases were washed with a saturated aqueous NaHCO3
(3×250 mL) and brine (3×250 mL), dried over Na2SO4,
and concentrated. The crude product was carried on directly to the next
step.

[0271] To the product from Step A (204 g, 750 mmol) was added CH3CN
(1500 mL). To this solution was added Selectfluor (292 g, 825 mmol)
portionwise (25 grams every 5 minutes) while cooling the reaction mixture
in a water bath. The reaction was stirred for 18 hours at ambient
temperature. The mixture was concentrated to dryness and the residue
dissolved in EtOAc (750 mL) and brine (500 mL). The organic layer was
washed with brine (250 mL) and dried over Na2SO4, filtered and
concentrated to dryness. The crude product was dissolved in minimal EtOAc
(150 mL) with heating until the solution was homogeneous and the solution
was allowed to cool to ambient temperature. Hexane (100 mL) was added
until the solution had become cloudy white. The mixture was allowed to
sit undisturbed for about 12 hours and the resulting solid isolated by
filtration. This provided 99 g (56%) of the product as a white solid.

[0272] To a slurry of NaBH4 (7.567 g, 200.0 mmol) in DCE (200 mL) was
added 2-Ethylhexanoic acid (95.50 mL, 600.0 mmol) slowly over 30 minutes
via addition funnel. The mixture was stirred at ambient temperature for 4
hours with venting to release H2. In a separate 1 L flask was added
the product from Step B (23.53 g, 100 mmol), benzylamine (16.37 mL, 150.0
mmol) and DCE (400 mL). The hydride solution was then added via addition
funnel to the mixture over 1 hours while cooling the reaction in a water
bath. The reaction was stirred at ambient temperature for 2 days, then
diluted with water (100 mL) and concentrated in vacuo to remove solvent.
The residue was partitioned between EtOAc (300 mL) and a saturated
aqueous Na2CO3 solution (2×75 mL). The mixture was
shaken, the layers separated and the organic phase washed again with a
saturated aqueous Na2CO3 solution (100 mL) and finally with
brine (50 mL). The aqueous phases were extracted with CHCl3
(3×75 mL), and the organic phases were dried over Na2CO3,
filtered and concentrated. The crude product was purified by column
chromatography (EtOAc/Hexane) providing 19.55 g of the pure cis product
and 5.0 g of a cis/trans mixture (80%).

[0273] To a 250 mL glass Parr vessel was added the product from Step C
(14.8 g, 48.0 mmol) and EtOH (100 mL). Pearlman's Catalyst (4.72 g, 3.36
mmol) was added and the mixture was shaken in a Parr reactor under 40-45
psi H2 for 15 hours. The reaction was filtered through celite and
the celite was washed with EtOAc. The filtrate was concentrated and the
residue was dissolved in CH2Cl2 and filtered through celite,
and the celite was washed with CH2Cl2 and EtOAc. The filtrate
was concentrated to afford 10.2 g (92%) of the desired product as a thick
oil that slowly solidified to a white solid.

[0274] To a solution of the product from Step D (10 g, 45.8 mmol) in THF
(90 mL) and water (20 mL) was added K2CO3 (8.23 g, 59.6 mmol).
Once the solid was dissolved benzyl chloroformate (7.19 mL, 50.4 mmol)
was added. The reaction was stirred vigorously at ambient temperature for
5.0 hours. Once the reaction was complete by TLC the reaction was diluted
with EtOAc (100 mL) and water (20 mL). The layers were separated and the
organic layer was washed with brine, then dried over Na2SO4,
filtered and concentrated in vacuo. The product was purified by column
chromatography (EtOAc/hexane) to afford 12.9 g (80%) of a sticky, white
foam.

Step F: Preparation of cis-benzyl-3-fluoropiperidin-4-ylcarbamate

[0275] (cis)-tert-Butyl
4-(benzyloxycarbonylamino)-3-fluoropiperidine-1-carboxylate (7.5 g, 21.3
mmol) was weighed into a 500 mL 1 neck round bottom and dissolved in 200
mL of DCM, followed by addition of TFA (16.4 mL, 213 mmol) and stirring
at ambient temperature for 1 hour, at which time all bubbling had ceased
and the reaction appeared complete by TLC. The crude reaction was
concentrated in vacuo, followed by aqueous work-up with 2 N NaOH and DCM.
The combined organics were dried over Na2SO4, filtered and
concentrated in vacuo to afford
benzyl(cis)-3-fluoropiperidin-4-ylcarbamate (4.25 g, 16.8 mmol, 79.2%
yield) as a white solid.

[0277] To the product from Step G (0.185 g, 0.373 mmol) was added HCl
(1.86 mL, 11.2 mmol, 6.0M aqueous) and the solution was warmed to
90° C. and stirred for 0.5 hours. The mixture was cooled to
ambient temperature and diluted with water (10 mL) and with
CH2Cl2 (10 mL). The layers were mixed and separated and the
aqueous phase was treated with saturated aqueous Na2CO3 until
pH=10. The aqueous phase was extracted with CH2Cl2 (4×10
mL). The combined organic phase was washed with water and brine, dried
over Na2SO4, filtered and concentrated. The crude residue was
purified by column chromatography (1 to 10% MeOH w/6%
NH4OH/CH2Cl2) to afford 0.094 g (69%) of the desired
product as a pale yellow/orange solid. MS ESI (+) m/z 363.2 (M+1)
detected.

Patent applications by Erik James Hicken, Boulder, CO US

Patent applications by Fredrik P. Marmsater, Boulder, CO US

Patent applications by John E. Robinson, Boulder, CO US

Patent applications by Joseph P. Lyssikatos, Piedmont, CA US

Patent applications by Mark C. Munson, Acton, MA US

Patent applications by Qian Zhao, El Cerrito, CA US

Patent applications by Robert Kirk Delisle, Lyons, CO US

Patent applications by Shelley Allen, Boulder, CO US

Patent applications by ARRAY BIOPHARMA INC.

Patent applications in class Ring nitrogen shared by two of the cyclos

Patent applications in all subclasses Ring nitrogen shared by two of the cyclos